Throughout this disclosure, various publications are referenced by first author and date, within parentheses, patent number or publication number. The complete bibliographic reference is given at the end of the application. The disclosures of these references are hereby incorporated by reference into this disclosure to more fully describe the state of the art to which this application pertains.
Enzyme Catalyzed Therapeutic Activation (ECTA™) therapy is a novel technology that provides unique prodrug substrates for target enzymes. Unlike conventional therapies, ECTA prodrugs neither inhibit nor irreversibly inactivate the target enzyme. U.S. Pat. Nos. 6,159,706; 6,245,750 and 6,339,151B1. See also PCT/US98/16607; PCT/US99/01332; and PCT/US00/20008.
Target enzymes convert the ECTA prodrug into a toxin preferentially within the target cell or in an environment wherein the target enzyme is expressed as compared to an environment where it is absent, as in an infected cell. Because the compounds do not require a targeting agent, they can be directly utilized, topically or systemically.
ECTA molecules do not, in most instances, yield cytotoxic products spontaneously (without target enzyme activation). They are not be appreciably activated by non-targeted enzymes, as this may result in toxicity to non-diseased or non-infected tissue. Table 1 summarizes the characteristics of ECTA molecules and enzyme activators.
TABLE 1Characteristics of ECTA TargetEnzymesCharacteristics of ECTA ProdrugsInfectious Disease: Must be presentMust be able to get into cells (byonly in target cells (includingitself or as prodrug).diseased cells, bacteria, fungi,etc.). The enzyme should benecessary for continuedviability or pathogenicity.Must process a molecule thatAt least one of the products formedresembles the natural substrate (anfrom the enzymatic reaction mustECTA molecule) into cytotoxicbe cytotoxic. However, the ECTAspecies. The resemblance only hasremain in inactive form untilto be significant with respect to theactivated by the target enzyme.specificity of the enzyme/substrateThe compound must have a highinteraction and the ability of thedegree of specificity for the targetedenzyme to process the substrateenzyme, although conversion by aintracellularly into toxic species.non-targeted enzyme is acceptableif the product(s) are not cytotoxic.Must not be inactivated by theMust not inhibit or deactivate theECTA molecule, intermediate(s),targeted enzyme.or the product(s) of the reaction.
In cases of bacterial, viral and fungal infections in plants, people or agriculturally important animals, metabolic pathways being present in the pathogenic organisms, but absent in the host are a source of potential ECTA target enzymes. For example, some pathways, as well as the enzymes involved, have only been found in bacteria, fungi and plants and not in mammalian cells. One example is the synthesis of “essential” amino acids—amino acids that animals cannot synthesize and must ingest with food. Nelson and Cox (1972).
Another example is Peptide Deformylase (“PDF”, EC 3.5.1.88) which catalyses deformylation of N-terminal N-formyl methionine in a growing polypeptide chain. Meinnel (1999). The enzyme is present and active in bacteria (Meinnel et al, 1993), but has not been reported to be active in mammalian cells. Sequences homologous to bacterial PDF sequences have been recently found in mammals but their exact function is unknown. Giglione (2000a) and (2000b).
Because the enzyme is not active in humans it has been used as a target for antibacterial drugs, mostly PDF inhibitors. Dithiols can act as non-specific PDF inhibitors by coordination of sulfhydryl groups with the active site metal ion. Rajagopalan (1997). In case of 1,2- or 1,3-dithiols a slow extraction of the metal ion from the active site takes place. The formation of stable 5- or 6-membered rings, respectively, each containing two metal-sulfur bonds, accounts for this effect.
A rationally designed combinatorial library was used to select mechanism-based PDF inhibitors of the general structure HS—CH2—CH(Ra)—CONH—CH(Rb)—CONH—Rc. Wei et al. (2000a). The optimal inhibitor selected from the library possesses an n-Bu group as an Ra, Rb=—(CH2)3—NH—C(═NH)—NH2, and Rc is 2-naphthalene. This compound acts competitive PDF inhibitor with a Ki of 15 nM.
Jayasekera et al. (2000) describes a series of non-peptidic compounds structurally related to the known anticholesteremic thyropropic acid to inhibit E. coli PDF. Actinonin is reported to be a potent PDF inhibitor with activity in the subnanomolar Ki range. Chen (2000) and Guilloteau et al (2002).
Wei, et al. (2000a) describe that 5′-dipeptidyl derivatives of 5-fluorodeoxyuridine release a small molecule (5-fluorodeoxyuridine (5-F-dUrd)) upon PDF catalyzed deformylation. 5-F-dUrd formation was monitored in the reaction of the substrate catalyzed by purified PDF or E. coli crude lysates. The compound was marginally cytotoxic (IC50>100 μM) when applied to E. coli bacteria. Potency was not increased by increased expression of PDF in bacteria (using a PDF-overexpressing strain). The compound was slightly more effective (IC50=50 μM) against gram-positive microorgansims.
Additional inhibitors are described in Apfel et al. (2000), Apfel et al. (2001a), Apfel et al. (2001b), Clements et al. (2001), Durand et al. (1999), Chen et al. (2000), Yuan et al (2001), and Wise et al (2002)
However, a compound or agent that is not an inhibitor but rather selectively and effectively activated by PDF to a toxin has not been described. This invention satisfies this need and provides related advantages as well.